Frameshift variants in C10orf71 cause dilated cardiomyopathy in human, mouse, and organoid models

Research advances over the past 30 years have confirmed a critical role for genetics in the etiology of dilated cardiomyopathies (DCMs). However, full knowledge of the genetic architecture of DCM remains incomplete. We identified candidate DCM causal gene, C10orf71, in a large family with 8 patients with DCM by whole-exome sequencing. Four loss-of-function variants of C10orf71 were subsequently identified in an additional group of492 patients with sporadic DCM from 2 independent cohorts. C10orf71 was found to be an intrinsically disordered protein specifically expressed in cardiomyocytes. C10orf71-KO mice had abnormal heart morphogenesis during embryonic development and cardiac dysfunction as adults with altered expression and splicing of contractile cardiac genes. C10orf71-null cardiomyocytes exhibited impaired contractile function with unaffected sarcomere structure. Cardiomyocytes and heart organoids derived from human induced pluripotent stem cells with C10orf71 frameshift variants also had contractile defects with normal electrophysiological activity. A rescue study using a cardiac myosin activator, omecamtiv mecarbil, restored contractile function in C10orf71-KO mice. These data support C10orf71 as a causal gene for DCM by contributing to the contractile function of cardiomyocytes. Mutation-specific pathophysiology may suggest therapeutic targets and more individualized therapy.


Figure S1, Cardiac pathological result of the heart transplantation patient
The sections are Masson trichrome stains from the left ventricle in which muscle is red and diffuse interstitial collagen is blue.The white spaces adjacent to myocytes are an artifact of myocyte shrinkage away from interstitial connective tissue with the formalin fixation and routine processing.

Figure S9, Expression of C10orf71 in GTEx Portal
The red frame indicates the expression of C10orf71 in heart.m, month; y, year.*Partial patients under the age of 18 did not reach the standard for systolic dysfunction, which is defined as enlarged heart rather than DCM.

Figure S2 ,
Figure S2, The genetic structure of the C10orf71 and its surrounding regions A, The genes near C10orf71.B, Linkage disequilibrium relationship of SNPs within 50 kb of either side of C10orf71 calculated by Haploview software using CHB data as a reference.

Figure S3 ,
Figure S3, Truncated C10orf71 proteins expressed by C10orf71 with frameshift variants A, Sequencing validation of expression plasmids for C10orf71 with variants found in DCM patients.The blue shadows indicate the mutation sites.B, Western blotting using cell lysates of HEK293T cells transfected with C10orf71 expression plasmids.

Figure S4 ,
Figure S4, Construction of C10orf71 mutant hiPSCs A, The gRNA used in the construction.B and C, Sequencing validation of C10orf71 mutant hiPSCs.The red boxes indicate the positions of the insertion of one base pair (B) and deletion of 11 base pairs (C).

Figure S5 ,
Figure S5, Quantitative analysis of C10orf71 mRNA with frameshift variant The level of frameshift variant mRNA was assessed using qPCR (A) and RNA sequencing (B).Each dot in panel A represents one biological repeat.Data represent means ± SD. *P < 0.05 in Mann-Whitney test.The red box in panel B indicates the C10orf71 protein coding region.

Figure S6 ,
Figure S6, Structural prediction of C10orf71 protein in different databases

Figure S10 ,
Figure S10, Expression level of cardiac marker Tnnt2 during heart developmentEach dot represents one biological repeat.Data represent means ± SD.

Figure S11 ,
Figure S11, Expression level of mC10orf71 during myogenesisA, Expression level of mC10orf71 in a public data from GEO database (GDS587).B, Expression level of mC10orf71 during myogenesis of C2C12 cells.Each dot represents one biological repeat.Data represent means ± SD.

Figure S12 ,
Figure S12, Expression of C10orf71 and cardiac markers (TNNT2 and MYH7) in different types of cells in our single nucleus RNA sequencing data of human heart The red boxes indicate cardiomyocytes.

Figure S13 ,
Figure S13, Expression of C10orf71 and cardiac markers (TNNT2 and MYH7) in different types of cells in a published single cell sequencing database A, UMAP of this data.B, Violin plots of the target genes.The red boxes indicate ventricular-cardiomyocytes.

Figure S16 ,
Figure S16, H&E staining of hearts of mice at different times after birth

Figure S18 ,
Figure S18, Echocardiographic parameters of WT and KO male mice HR: heart rate.LVVOL-d: left ventricular volume at end-diastole.LVID-d: internal

Figure S19 ,
Figure S19, Echocardiographic parameters of WT and KO male mice aged 53~63 weeks n = 5 for WT; n = 8 for KO.Each dot represents one biological repeat.Data represent means ± SD. **P < 0.01 in t-test.

Figure S20 ,
Figure S20, Myocardial images of WT and KO male mice aged 4 months taken by transmission electron microscopy

Figure S22 ,
Figure S22, Cardiac function of WT, heterogynous, and homozygous male mice aged 7~8-months A, Representative ultrasound images for hearts with different genotypes.B, Heart rates for hearts with different genotypes.C, Echocardiographic parameters for hearts with different genotypes.n = 7-8 per group.Each dot represents one biological repeat.Data represent means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 in one-way ANOVA combined with post hoc Tukey's test.ns, no significance.

Figure S24 ,
Figure S24, Expression level for genes related to heart contraction and RNA splicing in adult WT and KO mice A-B Heatmap of genes related to heart contraction (A) and RNA splicing (B).Red arrows indicate the known DCM genes.C, qPCR validation of expressional changes of Rbm24, Nexn, Chrm2, Dsg2, Pln, and Sgcb in adult WT and KO mice (n = 12-14).Each dot represents one biological repeat.Data represent means ± SD. ***P < 0.001 in t-test.

Figure S25 ,
Figure S25, The proportion of alternative splicing types in response to mC10orf71 knockout

Figure S26 ,
Figure S26, Splicing changes of Tnnt2 in KO mice at E18.5 IGV view showing reads mapping to Tnnt2.Due to significant differences in the transcription levels between exons in front and in back of the gene, the results are presented in two parts.

Figure S27 ,
Figure S27, Heatmap of expression values for genes related to extracellular matrix

Figure S36 ,
Figure S36, Upregulated GO terms after OM treatment (A) GO enrichment analysis of up-regulated genes after OM treatment (n = 4 per group).The top eight terms are listed.The red dots indicate the terms associated with CM differentiation and contraction.(B) Heatmap of expression values for contractile genes down-regulated in KO heart and up-regulated by OM treatment.(C) Heatmap of expression values for up-regulated contractile genes apart from genes in B. (D-F) Heatmap of expression values for genes related to fatty acid metabolism (D), Ca 2+ cycling/SR (E), and ion channels (F).

Figure S37 ,
Figure S37, Downregulated GO terms after OM treatment The top eight terms are listed.The green squares indicate the terms associated with catabolic process (n = 4 per group).